Translocation through TonB-dependent transporters E. coli bacteria, as well as pathogens such as Yersinia pestis, acquire essential nutrients such as iron and vitamin B12 through a group of ligand-gated outer membrane receptor proteins called TonB-dependent transporters. These receptors share a common structure; they are all 22-stranded b-barrels with a plug that faces the inside of the outer membrane. Nutrient uptake through these receptors depends on energy transduction from the inner membrane via the TonB protein, which makes contact with a sequence called the TonB box on the plug domain of the receptor, to move it out of the way and allow substrate entry. Protein toxins called colicins that are made by some E. coli to kill other E. coli have co-opted a number of TonB-dependent transporters as their outer membrane receptors, to attach to target bacteria. They must then cross the outer membrane, in a process called translocation, to deliver their toxic domain inside the target cell. It was recently discovered that colicin Ia uses its TonB-dependent outer membrane receptor, Cir, not just as its primary receptor, to attach to the cell surface, but also as its translocator across the membrane. The interaction of the colicin with Cir that initiates translocation is via its N-terminal translocation, or T, domain, rather than via the better-characterized interaction with the colicin's receptor-binding domain. This proposal is aimed at using the translocation domain of the colicin as a probe to identify the minimal signals that lead to the opening or unplugging of the Cir receptor in response to T domain binding, a process believed to mimic the opening of the receptors by their natural substrates. The minimal translocation peptide domain(s) that bind to the receptor will be identified by deletion analysis and isothermal calorimetry binding measurements. Mutant translocation-domain peptides will be used to freeze the translocation process at various steps, with cross-linking of binding partners, in order to better understand the entire process by which the colicin binds to its translocator and triggers its opening. PUBLIC HEALTH RELEVANCE: The TonB-dependent receptors used for colicin uptake are used by virtually all Gram-negative bacteria, including pathogens like Yersinia pestis, to acquire iron, essential to their viability. Better understanding the mechanisms that mobilize that uptake will allow design of means to inhibit that essential process.